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J.A. Franklyn, M. Wilson, J.R. Davis, D.B. Ramsden, K. Docherty and M.C. Sheppard

ABSTRACT

We have reported previously the effect of thyroid status in vivo on pituitary cytoplasmic concentrations of messenger RNA (mRNA) encoding the thyrotrophin (TSH) β-subunit (Franklyn, Lynam, Docherty et al, 1985). Studies in vitro of the regulation of TSH β gene transcription have been confined to thyrotrophic tumour cells. We now report the demonstration of TSH β-subunit mRNA in non-tumorous rat pituitary cells in primary culture. Treatment of cells with thyrotrophin-releasing hormone (TRH) and with forskolin resulted in a marked increase in cellular concentration of TSH β-mRNA. These results suggest that TRH exerts a direct effect on the pretranslational events involved in TSH synthesis and further that the adenylate cyclase system may be involved in the regulation of synthesis. We have thus described a novel system for the study of TSH β-subunit gene expression in normal rat pituitary cells in vitro.

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J J Whyte, A P Alexenko, A M Davis, M R Ellersieck, E D Fountain and C S Rosenfeld

We examined the effects of three maternal diets (very high fat (VHF), low fat (LF), and control (Purina 5015)) on serum steroids, free fatty acids (FFA), and vaginal pH in National Institutes of Health Swiss mice. Females were fed (VHF, n = 33; LF, n = 33; 5015, n = 48) from 4 to 16 weeks of age. Following breeding, female serum was collected at 0.5 (pre-implantation, early diestrus) or 8.5 (post-implantation, mid-diestrus) days post-coitus (dpc). The serum concentrations of 17β-estradiol, testosterone, progesterone, and FFA were analyzed at both collection points, and vaginal pH at 0.5 dpc. Striking differences in steroids and FFA were observed at 0.5 dpc among the groups. Estradiol was higher in the VHF (14.1 ± 3.0 pg/ml), compared with LF mice (5.2 ± 2.3 pg/ml; P≤ 0.05). In contrast, 0.5 dpc testosterone was lower in the VHF (10.5 ± 3.0 pg/ml) versus the LF group (32.7 ± 8.4 pg/ml; P≤ 0.05). At 8.5 dpc, progesterone was higher in the VHF (89.6 ± 6.7 ng/ml) versus the 5015 group (60.1 ± 4.9 ng/ml; P≤ 0.05). VHF mice had higher FFA concentrations at 0.5 dpc (1.0 ± 0.2 mmol/l) than LF and control mice (0.5 ± 0.1 and 0.6 ± 0.1 mmol/l respectively; P≤ 0.05). At 8.5 dpc, VHF females had higher serum FFA (0.8 ± 0.1 mmol/l) than LF and control females (0.4 ± 0.1 and 0.6 ± 0.1 mmol/l; P≤ 0.05). Mean vaginal pH of VHF females (6.41 ± 0.09) was lower than 5015 females (6.76 ± 0.10; P≤ 0.05). These diet-induced alterations in serum steroid and FFA concentrations might affect several reproductive processes, including preferential fertilization by one class of sperm over the other and sex bias in pre- and post-implantational embryonic development.

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J. R. E. Davis, T. C. Lynam, J. A. Franklyn, K. Docherty and M. C. Sheppard

ABSTRACT

Thyroid hormones may regulate prolactin gene transcription. We have previously found that phenytoin inhibits tri-iodothyronine (T3) nuclear binding, and have suggested that phenytoin may act as a partial T3 agonist. We have therefore investigated the effects of phenytoin and T3 on prolactin release and gene transcription, using the technique of cytoplasmic dot hybridization with complementary DNA probes to estimate prolactin messenger (m) RNA concentrations in cytoplasm from cultured rat pituitary cells.

Tri-iodothyronine treatment led to a small but significant fall in prolactin release by 72 h, but caused marked dose- and time-dependent reductions in prolactin mRNA levels at 48–72 h. Phenytoin, however, caused more rapid falls in both prolactin release and mRNA concentrations. Neither T3 nor phenytoin significantly altered GH mRNA levels. These studies suggest effects of phenytoin similar, but not identical, to those of T3 in the lactotroph.

J. Endocr. (1986) 109, 359–364

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E. G. Black, A. Logan, J. R. E. Davis and M. C. Sheppard

ABSTRACT

We have used a recombinant human basic fibroblast growth factor (basic FGF) to study its effects on cell proliferation, gene expression and accumulation of cyclic AMP (cAMP) and inositol phosphates in two well-characterized endocrine cell lines, FRTL-5 rat thyroid and GH3 rat pituitary cells. Basic FGF induced a dose-dependent increase in mitogenesis (assessed by measuring incorporation of [3H]thymidine) in FRTL-5 cells (40 ng basic FGF/ml increased mitogenesis above the control value by 2148±108% (mean ± s.e.m.), but inhibited mitogenesis in GH3 cells at all doses (85±4% of control with 40 ng basic FGF/ml)). Thyroglobulin mRNA concentration was increased in FRTL-5 cells (126±6% of control with 40 ng basic FGF/ml) as was prolactin mRNA in GH3 cells (246±11% of control with 40 ng basic FGF/ml), but GH mRNA in GH3 cells was not significantly affected by any dose of basic FGF. Intracellular cAMP was reduced by basic FGF in both FRTL-5 and GH3 cells (40 ng bFGF/ml giving 80±5% of the control value in FRTL-5, and 67±15% of the control value in GH3 cells) despite increased levels when FRTL-5 cells were stimulated with 150 μU TSH/ml (5645±484% of control) or GH3 cells were stimulated by 10 μmol forskolin/1 (3347±396% of control). In both FRTL-5 and GH3 cells, accumulation of [3H]inositol phosphates was increased by 40 ng basic FGF/ml (201±6 and 330±51% of control values respectively).

We have shown that basic FGF has different effects on mitogenesis in the two cell lines; gene expression and accumulation of inositol phosphates were increased in both, whereas the intracellular concentration of cAMP was decreased. The actions of basic FGF may be mediated through both inhibition of adenylate cyclase and hydrolysis of phosphatidyl inositol bisphosphate as has been proposed for 3T3 fibroblasts. Our data suggest that there may be a physiological role for basic FGF in both thyroid and pituitary tissue.

Journal of Endocrinology (1990) 127, 39–46

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J. A. Franklyn, J. R. E. Davis, D. B. Ramsden and M. C. Sheppard

ABSTRACT

Circulating free thyroid hormone concentrations are reduced in subjects taking long-term phenytoin, a finding at variance with their euthyroid clinical state and normal serum TSH concentration. It is suggested, therefore, that phenytoin may modify the cellular effects of thyroid hormones.

In order to examine the influence of phenytoin on thyroid hormone action in the pituitary gland we studied its effect on the binding of tri-iodothyronine (T3) to isolated nuclei prepared from rat anterior pituitary tissue. Phenytoin inhibited the nuclear binding of T3 in a dose-dependent fashion. Phenytoin also partially inhibited thyrotrophin-releasing hormone-stimulated TSH release from cultured rat anterior pituitary cells. These studies provide evidence for a direct effect of phenytoin on the thyrotroph mediated via nuclear T3 receptor binding.

J. Endocr. (1985) 104, 201–204

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C G Prosser, S R Davis, S C Hodgkinson and M A Mohler

Abstract

The aim of this study was to compare the plasma concentration profile, mammary blood flow response and transfer into milk of intact IGF-I with that of its truncated analogue, des(1–3)IGF-I (des-IGF-I). Each peptide was infused for 24 h into the pudic artery supplying one mammary gland of lactating goats (n=5). Concentrations of IGF-I in plasma (from the jugular vein) rose rapidly during infusion of IGF-I or des-IGF-I to reach 510±62 and 640±32 ng/ml (mean ± s.e.m.) respectively, compared with 262±35 ng/ml after a similar infusion of saline. Ligand blotting analysis indicated a significant increase in the intensity of [125I]IGF-I binding to the 40–43 kDa doublet (binding protein-3 (BP-3), P<0·01) and the band at 31 kDa (P<0·05) during infusion of either IGF-I or des-IGF-I, as compared with saline. Furthermore des-IGF-I induced a significant increase in intensity of binding to the 35 and 24 kDa bands, but IGF-I did not. Whereas [125I]IGF-I was distributed between BP-3 and the other binding proteins, [125I]des-IGF-I bound exclusively to BP-3.

Mammary blood flow (MBF) increased 48±6% after 12 h of infusion of des-IGF-I, compared with an increase of 22±6% during IGF-I. The difference in response was significant at P<0·05. In addition, more IGF-I was secreted into the milk of the infused than the non-infused gland during either infusion of IGF-I or des-IGF-I. This difference between glands was greater (P<0·05) during des-IGF-I compared with IGF-I infusion, suggesting greater uptake of des-IGF-I by the gland compared with IGF-I, when infused locally. These findings indicate a greater bioactivity of des-IGF-I compared with IGF-I when infused locally into the mammary gland and may be explained by the different pattern of association of the two peptides with different binding proteins. The similar plasma profile and pharmacokinetics for IGF-I and desIGF-I during their 24-h continuous infusion of des-IGF-I or IGF-I is in contrast to results reported for a single injection of the peptides, probably relating to the ability of des-IGF-I to induce and bind to BP-3.

Journal of Endocrinology (1995) 144, 99–107

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R. B. Heap, I. R. Fleet, A. J. Davis, J. A. Goode, M. H. Hamon, D. E. Walters and A. P. F. Flint

ABSTRACT

The mechanisms of lymphatic-vascular transfer across the ovarian vascular pedicle were studied in anaesthetized sheep 8–15 days after ovulation. [3H]Prostaglandin F (PGF), [14C]mannitol and [36Cl]Na were infused continuously into either a uterine lymphatic or a uterine vein and the kinetics of transfer into the adjacent utero-ovarian vein or ovarian plasma were studied. Transfer occurred according to the sequence [36Cl] > [14C] > [3H] indicating that PGF is not transferred by rapid diffusion, as with [36Cl]Na, nor by a paracellular route, as with [14C]mannitol, but by a slower process probably involving facilitated diffusion.

Transfer into the adjacent utero-ovarian vein or ovarian blood was greater when compounds were infused into a uterine lymphatic than into a uterine vein. Substantially more [3H]PGF occurred in the adjacent corpus luteum than either of the other compounds after a lymphatic infusion. Intra-lymphatic infusion of PGF stimulated the release of ovarian oxytocin but the effect was not confined to the adjacent ovary. Intravenous (jugular) infusion of PGF failed to stimulate ovarian oxytocin secretion whereas close-arterial infusion into the ovaries was effective, and the possibility was investigated that any systemic effect of PGF was mediated through neural mechanisms. Noradrenaline and acetylcholine were both effective in causing the release of ovarian oxytocin when infused close-arterially into the ovary. With infusions of acetylcholine, ovarian oxytocin secretion rate was increased over fivefold without any change in posterior pituitary release. Noradrenaline and acetylcholine produced a concomitant fall in ovarian blood flow, and neurotransmitter-induced ischaemia may have played a role in ovarian oxytocin release. The finding that PGF infused into a uterine lymphatic stimulates ovarian secretion of oxytocin, and that the effect is bilateral whereas PGF accumulation in ovarian tissue is unilateral, implies that its mechanism of action may not be solely directed at the luteal cell.

Journal of Endocrinology (1989) 122, 147–159

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T J Kowalski, B D Spar, L Markowitz, M Maguire, A Golovko, S Yang, C Farley, J A Cook, G Tetzloff, L Hoos, R A Del Vecchio, T M Kazdoba, M F McCool, J J Hwa, L A Hyde, H Davis, G Vassileva, J A Hedrick and E L Gustafson

Recent work has shown that neuromedin U (NmU), a peptide initially identified as a smooth muscle contractor, may play a role in regulating food intake and energy homeostasis. To further evaluate this putative function, we measured food intake, body weight, energy expenditure and glucose homeostasis in transgenic mice that ubiquitously overexpress murine proNmU. NmU transgenic mice were lighter and had less somatic and liver fat, were hypophagic, and had improved insulin sensitivity as judged by an intraperitoneal insulin tolerance test. Transgenic mice had higher levels of hypothalamic NPY, POMC and MCH mRNA. There was no difference in O2 consumption between genotypes; however, NmU transgenic mice displayed a modest increase in respiratory quotient during food deprivation and refeeding. There were no behavioral disturbances in the NmU transgenic mice that could account for the results (e.g. changes in locomotor activity). When placed on a high-fat diet, transgenic mice remained lighter than wild-type mice and ate less, but gained weight at a rate similar to wild-type mice. Despite the increased weight gain with high-fat feeding, glucose tolerance was significantly improved in the transgenic mice. These findings support the hypothesized role of NmU as an endogenous anorexigenic peptide.